Corn event PV-ZMGT32(nk603) and compositions and methods for detection thereof

ABSTRACT

The present invention provides a DNA construct that confers tolerance to transgenic corn plant. Also provided are assays for detecting the presence of the PV-ZMGT32(nk603) corn event based on the DNA sequence of the recombinant construct inserted into the corn genome and of genomic sequences flanking the insertion site.

FIELD OF THE INVENTION

The present invention relates to the field of plant molecular biology,specifically the invention relates to a DNA construct for conferringglyphosate tolerance to a plant. The invention more specifically relatesto a glyphosate tolerant corn plant PV-ZMGT32(nk603) and to assays fordetecting the presence of corn plant PV-ZMGT32(nk603) DNA in a sampleand compositions thereof.

BACKGROUND OF THE INVENTION

This invention relates to the glyphosate herbicide tolerant corn (Zeamays) plant PV-ZMGT32(nk603) and to the DNA plant expression constructof corn plant PV-ZMGT32(nk603) and the detection of thetransgene/genomic insertion region in corn PV-ZMGT32(nk603) and progenythereof.

Corn is an important crop and is a primary food source in many areas ofthe world. The methods of biotechnology have been applied to corn forimprovement of the agronomic traits and the quality of the product. Onesuch agronomic trait is herbicide tolerance, in particular, tolerance toglyphosate herbicide. This trait in corn has been conferred by theexpression of a transgene in the corn plants (U.S. Pat. No. 6,040,497).

The expression of foreign genes in plants is known to be influenced bytheir chromosomal position, perhaps due to chromatin structure (e.g.,heterochromatin) or the proximity of transcriptional regulation elements(e.g., enhancers) close to the integration site (Weising et al., Ann.Rev. Genet 22:421-477, 1988). For this reason, it is often necessary toscreen a large number of events in order to identify an eventcharacterized by optimal expression of a introduced gene of interest.For example, it has been observed in plants and in other organisms thatthere may be a wide variation in levels of expression of an introducedgenes among events. There may also be differences in spatial or temporalpatterns of expression, for example, differences in the relativeexpression of a transgene in various plant tissues, that may notcorrespond to the patterns expected from transcriptional regulatoryelements present in the introduced gene construct. For this reason, itis common to produce hundreds to thousands of different events andscreen those events for a single event that has desired transgeneexpression levels and patterns for commercial purposes. An event thathas desired levels or patterns of transgene expression is useful forintrogressing the transgene into other genetic backgrounds by sexualoutcrossing using conventional breeding methods. Progeny of such crossesmaintain the transgene expression characteristics of the originaltransformant. This strategy is used to ensure reliable gene expressionin a number of varieties that are well adapted to local growingconditions.

It would be advantageous to be able to detect the presence of aparticular event in order to determine whether progeny of a sexual crosscontain a transgene of interest. In addition, a method for detecting aparticular event would be helpful for complying with regulationsrequiring the premarket approval and labeling of foods derived fromrecombinant crop plants, for example. It is possible to detect thepresence of a transgene by any well known nucleic acid detection methodsuch as the polymerase chain reaction (PCR) or DNA hybridization usingnucleic acid probes. These detection methods generally focus onfrequently used genetic elements, such as promoters, terminators, markergenes, etc. As a result, such methods may not be useful fordiscriminating between different events, particularly those producedusing the same DNA construct unless the DNA sequence of chromosomal DNAadjacent to the inserted DNA (“flanking DNA”) is known. Anevent-specific PCR assay is discussed, for example, by Windels et al.(Med. Fac. Landbouww, Univ. Gent 64/5b:459-462, 1999), who identifiedglyphosate tolerant soybean event 40-3-2 by PCR using a primer setspanning the junction between the insert and flanking DNA, specificallyone primer that included sequence from the insert and a second primerthat included sequence from flanking DNA.

SUMMARY OF THE INVENTION

According to one aspect of the invention, a DNA construct is providedthat when expressed in plant cells and plants confers tolerance toglyphosate herbicide. This invention relates preferably to the methodsfor producing and selecting a glyphosate tolerant monocot crop plant.The DNA construct consists of two transgene expression cassettes. Thefirst expression cassette comprising a DNA molecule of a rice (Oryzaesativa) actin 1 promoter and rice actin 1 intron operably joined to aDNA molecule encoding a chloroplast transit peptide sequence, operablyconnected to a DNA molecule encoding a glyphosate resistant5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS), operably connectedto a DNA molecule comprising a 3′ transcriptional terminator. The secondtransgene expression cassette of the DNA construct comprising a DNAmolecule of the cauliflower mosaic virus (CaMV) 35S promoter, operablyconnected to a DNA molecule comprising a Hsp70 intron, operablyconnected to a DNA molecule encoding a chloroplast transit peptidesequence, operably connected to a DNA molecule encoding a glyphosateresistant 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS), operablyconnected to a DNA molecule comprising a 3′ transcriptional terminator.

More specifically, a DNA construct is provided that when expressed inplant cells and plants confers tolerance to glyphosate herbicide. Thisinvention relates preferably to the methods for producing and selectinga glyphosate tolerant corn plant. The DNA construct consists of twotransgene expression cassettes. The first expression cassette consistingof a DNA molecule of a rice (Oryzae sativa) actin 1 promoter and riceactin 1 intron operably joined to a DNA molecule encoding an ArabidopsisEPSPS chloroplast transit peptide sequence, operably connected to a DNAmolecule encoding a glyphosate resistant5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) isolated fromAgrobacterium tumefaciens sp. strain CP4, operably connected to a DNAmolecule consisting of a nopaline synthase transcriptional terminator.The second transgene expression cassette consisting of a DNA molecule ofthe cauliflower mosaic virus (CaMV) 35S promoter containing a tandemduplication of the enhancer region, operably connected to a DNA moleculeconsisting of a Zea mays Hsp70 intron, operably connected to a DNAmolecule encoding an Arabidopsis EPSPS chloroplast transit peptidesequence, operably connected to a DNA molecule encoding a glyphosateresistant 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) isolatedfrom Agrobacterium tumefaciens sp. strain CP4, operably connected to aDNA molecule consisting of a nopaline synthase transcriptionalterminator.

According to another aspect of the invention, compositions and methodsare provided for detecting the presence of the transgene/genomicinsertion region from a novel corn plant designated PV-ZMGT32(nk603).DNA molecules are provided that comprise at least one junction sequenceof PV-ZMGT32(nk603) selected from the group consisting of 5′TGTAGCGGCCCACGCGTGGT 3′ (SEQ ID NO:9), 5′ TACCACGCGACACACTTC 3′ (SEQ IDNO: 10), and 5′ TGCTGTTCTGCTGACTTT 3′ (SEQ ID NO:11) and complementsthereof; wherein a junction sequence spans the junction betweenheterologous DNA inserted into the genome and the DNA from the corn cellflanking the insertion site and is diagnostic for the event. The cornplant and seed comprising these molecules is an aspect of thisinvention.

A novel DNA molecule 5′ACCAAGCTTTTATAATAG 3′ (SEQ ID NO:12) and thecomplement thereof, wherein this DNA molecule is novel inPV-ZMGT32(nk603) and its progeny. The corn plant and seed comprisingthis molecule is an aspect of this invention.

According to another aspect of the invention, DNA molecules thatcomprise the novel transgene/genomic insertion region, SEQ ID NO:7 andSEQ ID NO:8 and are homologous or complementary to SEQ NO:7 and SEQ IDNO:8 are an aspect of this invention.

DNA molecules that comprise a sufficient length of a transgene portionof the DNA sequence of SEQ ID NO:7 and a similar sufficient length of a5′ flanking corn DNA sequence of SEQ ID NO:7; or a similar sufficientlength of a transgene portion of the DNA sequence of SEQ ID NO:8 and asimilar sufficient length of a 3′ DNA sequence flanking the transgene,wherein these DNA molecules are useful as DNA primers in DNAamplification methods so as to provide a DNA amplicon productspecifically produced from PV-ZMGT32(nk603) DNA and its progeny areanother aspect of the invention. DNA primers homologous or complementaryto a length of SEQ ID NO:7 and SEQ ID NO:8 are an aspect of theinvention. The amplicons produced using DNA primers that are diagnosticfor corn event PV-ZMGT32(nk603) and its progeny are a subject of thisinvention.

According to another aspect of the invention, methods of detecting thepresence of DNA corresponding to the corn event PV-ZMGT32(nk603) eventin a sample are provided. Such methods comprise: (a) contacting thesample comprising DNA with a DNA primer set, that when used in a nucleicacid amplification reaction with genomic DNA extracted from corn eventPV-ZMGT32 (nk603) produces an amplicon that is diagnostic for corn eventPV-ZMGT32(nk603); (b) performing a nucleic acid amplification reaction,thereby producing the amplicon; and (c) detecting the amplicon. A pairof DNA molecules comprising a DNA primer set that are homologous orcomplementary to SEQ ID NO:7 or SEQ ID NO:8 that function in a nucleicacid amplification reaction to produce an amplicon DNA moleculediagnostic for PV-ZMGT329nk603). More specifically, a pair of DNAmolecules comprising a DNA primer set, wherein the DNA molecules areidentified as SEQ ID NO: 13 or complements thereof and SEQ ID NO: 14 orcomplements thereof; SEQ ID NO: 15 or complements thereof and SEQ ID NO:16 or complements thereof. The amplicon comprising the DNA molecules ofSEQ ID NO: 13 and SEQ ID NO: 14. The amplicon comprising the DNAmolecules of SEQ ID NO: 15 and SEQ ID NO: 16. The amplicon produce bythe afore described method that can hybridize under stringent conditionsto SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, or SEQ ID NO: 12.

According to another aspect of the invention, methods of detecting thepresence of a DNA molecule corresponding to the PV-ZMGT32(nk603) eventin a sample, such methods comprising: (a) contacting the samplecomprising DNA extracted from a corn plant with a DNA probe moleculethat hybridizes under stringent hybridization conditions with genomicDNA from corn event PV-ZMGT32(nk603) and does not hybridize under thestringent hybridization conditions with a control corn plant DNA; (b)subjecting the sample and probe to stringent hybridization conditions;and (c) detecting hybridization of the probe to the DNA. Morespecifically, a method for detecting the presence of a DNA moleculecorresponding to the PV-ZMGT32(nk603) event in a sample, such methods,consisting of (a) contacting the sample comprising DNA extracted from acorn plant with a DNA probe molecule that consists of SEQ ID NO:9, SEQID NO: 10, SEQ ID NO: 1, or SEQ ID NO: 12, wherein said DNA probemolecule hybridizes under stringent hybridization conditions withgenomic DNA from corn event PV-ZMGT32(nk603) and does not hybridizeunder the stringent hybridization conditions with a control corn plantDNA; (b) subjecting the sample and probe to stringent hybridizationconditions; and (c) detecting hybridization of the probe to the DNA.

According to another aspect of the invention, methods of producing acorn plant that tolerates application of glyphosate are provided thatcomprise the steps of: (a) sexually crossing a first parental corn linecomprising the expression cassettes of the present invention, whichconfers tolerance to application of glyphosate, and a second parentalcorn line that lacks the glyphosate tolerance, thereby producing aplurality of progeny plants; and (b) selecting a progeny plant thattolerates application of glyphosate. Such methods may optionallycomprise the further step of back-crossing the progeny plant to thesecond parental corn line to producing a true-breeding corn plant thattolerates application of glyphosate.

According to another aspect of the invention, a method is provided toselect for glyphosate tolerant corn plants of the present invention andprogeny thereof comprising extracting DNA from a plant sample,contacting a DNA with a marker nucleic acid molecule selected from thegroup consisting of SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ IDNO: 12, or complements thereof, detecting the hybridization of saidmarker nucleic acid molecule to the DNA, and performing a markerassisted breeding analysis for the genetic linkage of the glyphosatetolerant trait to the marker nucleic acid molecule.

The present invention provides a method of producing a corn planttolerant to glyphosate herbicide comprising transforming a corn cellwith the DNA construct (pMON25496), selecting the corn cell fortolerance to the treatment with an effective dose of glyphosate, andgrowing the corn cell into a fertile corn plant. The fertile corn plantcan be self pollinated or crossed with compatible corn varieties toproduce glyphosate tolerant progeny.

The invention further relates to a DNA detection kit comprising at leastone DNA molecule of sufficient length of contiguous nucleotideshomologous or complementary to SEQ ID NO:7 or SEQ ID NO:8 that functionsas a DNA primer or probe specific for corn event PV-ZMGT32(nk603) or itsprogeny.

This invention further relates to the plants and seeds of glyphosatetolerant corn (Zea mays) PV-ZMGT32 (nk603) having ATCC Accession No.PTA-2478 and the progeny derived thereof. The corn plant or its partsproduced by growing of the glyphosate tolerant corn plantPV-ZMGT32(nk603), the pollen and ovules of the corn plantPV-ZMGT32(nk603). The nuclei of vegetative cells, the nuclei of pollencells, and the nuclei of egg cells of the corn plant PV-ZMGT32 (nk603)and the progeny derived thereof. The corn plant and seedPV-ZMGT32(nk603) from which the DNA primer molecules of the presentinvention provide a specific amplicon product is an aspect of theinvention.

The foregoing and other aspects of the invention will become moreapparent from the following detailed description and accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Plasmid map of pMON25496

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This application claims the benefit of U.S. Provisional Application No.60/213,567, filed Jun. 22, 2000; U.S. Provisional Application No.60/241,215, filed Oct. 13, 2000; and U.S. Provisional Application No.60/240,014, filed Oct. 13, 2000. The following definitions and methodsare provided to better define the present invention and to guide thoseof ordinary skill in the art in the practice of the present invention.Unless otherwise noted, terms are to be understood according toconventional usage by those of ordinary skill in the relevant art.Definitions of common terms in molecular biology may also be found inRieger et al., Glossary of Genetics: Classical and Molecular, 5thedition, Springer-Verlag: New York, 1991; and Lewin, Genes V, OxfordUniversity Press: New York, 1994. The nomenclature for DNA bases as setforth at 37 CFR § 1.822 is used.

As used herein, the term “corn” means Zea mays or maize and includes allplant varieties that can be bred with corn, including wild maizespecies.

As used herein, the term “comprising” means “including but not limitedto”.

“Glyphosate” refers to N-phosphonomethylglycine and its salts,Glyphosate is the active ingredient of Roundup® herbicide (MonsantoCo.). Treatments with “glyphosate herbicide” refer to treatments withthe Roundup®, Roundup Ultra®, Roundup UltraMax® herbicide or any otherherbicide formulation containing glyphosate. The selection ofapplication rates for a glyphosate formulation that constitute abiologically effective dose is within the skill of the ordinaryagricultural technician.

A DNA construct is an assembly of DNA molecules linked together thatprovide one or more expression cassettes. The DNA construct ispreferably a plasmid that is enabled for self replication in a bacterialcell and contains various endonuclease enzyme restriction sites that areuseful for introducing DNA molecules that provide functional geneticelements, i.e., promoters, introns, leaders, coding sequences, 3′termination regions, among others. The expression cassettes containedwithin a DNA construct comprise the necessary genetic elements toprovide transcription of a messenger RNA. The expression cassettes canbe designed to express in prokaryote cells or eukaryotic cells. Theexpression cassettes of the present invention are designed to expressmost preferably in plant cells.

A transgenic “event” is produced by transformation of plant cells withheterologous DNA construct, including a nucleic acid expression cassettethat comprises a transgene of interest, the regeneration of a populationof plants resulting from the insertion of the transgene into the genomeof the plant, and selection of a particular plant characterized byinsertion into a particular genome location. The term “event” refers tothe original transformant and progeny of the transformant that includethe heterologous DNA. The term “event” also refers to progeny producedby a sexual outcross between the transformant and another variety thatinclude the heterologous DNA. Even after repeated back-crossing to arecurrent parent, the inserted DNA and flanking DNA from the transformedparent is present in the progeny of the cross at the same chromosomallocation. The term “event” also refers to DNA from the originaltransformant comprising the inserted DNA and flanking genomic sequenceimmediately adjacent to the inserted DNA that would be expected to betransferred to a progeny that receives inserted DNA including thetransgene of interest as the result of a sexual cross of one parentalline that includes the inserted DNA (e.g., the original transformant andprogeny resulting from selfing) and a parental line that does notcontain the inserted DNA. A glyphosate tolerant corn plant PV-ZMGT32(nk603) can be breed by first sexually crossing a first parental cornplant consisting of a corn plant grown from the transgenic corn plantPV-ZMGT32 (nk603) having ATCC Accession No. PTA-2478 and progeny thereofderived from transformation with the expression cassettes of the presentinvention that tolerates application of glyphosate herbicide, and asecond parental corn plant that lacks the tolerance to glyphosateherbicide, thereby producing a plurality of first progeny plants; andthen selecting a first progeny plant that is tolerant to application ofglyphosate herbicide; and selfing the first progeny plant, therebyproducing a plurality of second progeny plants; and then selecting fromthe second progeny plants a glyphosate herbicide tolerant plant. Thesesteps can further include the back-crossing of the first glyphosatetolerant progeny plant or the second glyphosate tolerant progeny plantto the second parental corn plant or a third parental corn plant,thereby producing a corn plant that tolerates the application ofglyphosate herbicide.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating added, exogenous genes. Selfing of appropriate progeny canproduce plants that are homozygous for both added, exogenous genes.Back-crossing to a parental plant and out-crossing with a non-transgenicplant are also contemplated, as is vegetative propagation. Descriptionsof other breeding methods that are commonly used for different traitsand crops can be found in one of several references, e.g., Fehr, inBreeding Methods for Cultivar Development, Wilcox J. ed., AmericanSociety of Agronomy, Madison Wis. (1987).

A “probe” is an isolated nucleic acid to which is attached aconventional detectable label or reporter molecule, e.g., a radioactiveisotope, ligand, chemiluminescent agent, or enzyme. Such a probe iscomplementary to a strand of a target nucleic acid, in the case of thepresent invention, to a strand of genomic DNA from corn eventPV-ZMGT32(nk603) whether from a corn plant or from a sample thatincludes DNA from the event. Probes according to the present inventioninclude not only deoxyribonucleic or ribonucleic acids but alsopolyamides and other probe materials that bind specifically to a targetDNA sequence and can be used to detect the presence of that target DNAsequence.

“Primers” are isolated nucleic acids that are annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, then extended alongthe target DNA strand by a polymerase, e.g., a DNA polymerase. Primerpairs of the present invention refer to their use for amplification of atarget nucleic acid sequence, e.g., by the polymerase chain reaction(PCR) or other conventional nucleic-acid amplification methods.

Probes and primers are of sufficient nucleotide length to bind to thetarget DNA sequence specifically in the hybridization conditions orreaction conditions determined by the operator. This length may be ofany length that is of sufficient length to be useful in the detectionmethod of choice. Generally, 11 nucleotides or more in length,preferably 18 nucleotides or more, more preferably 24 nucleotides ormore, and most preferably 30 nucleotides or more are used. Such probesand primers hybridize specifically to a target sequence under highstringency hybridization conditions. Preferably, probes and primersaccording to the present invention have complete DNA sequence similarityof contiguous nucleotides with the target sequence, although probesdiffering from the target DNA sequence and that retain the ability tohybridize to target DNA sequences may be designed by conventionalmethods.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3,ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989 (hereinafter, “Sambrook et al., 1989”); CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates)(hereinafter, “Ausubel et al., 1992”); and Innis et al., PCR Protocols:A Guide to Methods and Applications, Academic Press: San Diego, 1990.PCR-primer pairs can be derived from a known sequence, for example, byusing computer programs intended for that purpose such as Primer(Version 0.5, @1991, Whitehead Institute for Biomedical Research,Cambridge, Mass.).

Primers and probes based on the flanking DNA and insert sequencesdisclosed herein can be used to confirm (and, if necessary, to correct)the disclosed sequences by conventional methods, e.g., by re-cloning andsequencing such sequences.

The nucleic acid probes and primers of the present invention hybridizeunder stringent conditions to a target DNA sequence. Any conventionalnucleic acid hybridization or amplification method can be used toidentify the presence of DNA from a transgenic event in a sample.Nucleic acid molecules or fragments thereof are capable of specificallyhybridizing to other nucleic acid molecules under certain circumstances.As used herein, two nucleic acid molecules are said to be capable ofspecifically hybridizing to one another if the two molecules are capableof forming an anti-parallel, double-stranded nucleic acid structure. Anucleic acid molecule is said to be the “complement” of another nucleicacid molecule if they exhibit complete complementarity. As used herein,molecules are said to exhibit “complete complementarity” when everynucleotide of one of the molecules is complementary to a nucleotide ofthe other. Two molecules are said to be “minimally complementary” ifthey can hybridize to one another with sufficient stability to permitthem to remain annealed to one another under at least conventional“low-stringency” conditions. Similarly, the molecules are said to be“complementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another underconventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook et al., 1989, and by Haymes et al.,In: Nucleic Acid Hybridization, A Practical Approach, IRL Press,Washington, D.C. (1985), Departures from complete complementarity aretherefore permissible, as long as such departures do not completelypreclude the capacity of the molecules to form a double-strandedstructure. In order for a nucleic acid molecule to serve as a primer orprobe it need only be sufficiently complementary in sequence to be ableto form a stable double-stranded structure under the particular solventand salt concentrations employed.

As used herein, a substantially homologous sequence is a nucleic acidmolecule that will specifically hybridize to the complement of thenucleic acid molecule to which it is being compared under highstringency conditions. Appropriate stringency conditions which promoteDNA hybridization, for example, 6.0× sodium chloride/sodium citrate(SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C., areknown to those skilled in the art or can be found in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Forexample, the salt concentration in the wash step can be selected from alow stringency of about 2.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C. Both temperature andsalt may be varied, or either the temperature or the salt concentrationmay be held constant while the other variable is changed. In a preferredembodiment, a nucleic acid of the present invention will specificallyhybridize to one or more of the nucleic acid molecules set forth in SEQID NO: 9, 10, 11 and 12 or complements thereof or fragments of eitherunder moderately stringent conditions, for example at about 2.0×SSC andabout 65° C. In a particularly preferred embodiment, a nucleic acid ofthe present invention will specifically hybridize to one or more of thenucleic acid molecules set forth in SEQ ID NO:9 through SEQ ID NO:12 orcomplements or fragments of either under high stringency conditions. Inone aspect of the present invention, a preferred marker nucleic acidmolecule of the present invention has the nucleic acid sequence setforth in SEQ ID NO:9 through SEQ ID NO: 12 or complements thereof orfragments of either. In another aspect of the present invention, apreferred marker nucleic acid molecule of the present invention sharesbetween 80% and 100% or 90% and 100% sequence identity with the nucleicacid sequence set forth in SEQ ID NO:9 through SEQ ID NO:12 orcomplement thereof or fragments of either. In a further aspect of thepresent invention, a preferred marker nucleic acid molecule of thepresent invention shares between 95% and 100% sequence identity with thesequence set forth in SEQ ID NO:9 through SEQ ID NO: 12 or complementthereof or fragments of either. SEQ ID NO:9 through SEQ IN NO:12 may beused as markers in plant breeding methods to identify the progeny ofgenetic crosses similar to the methods described for simple sequencerepeat DNA marker analysis, in “DNA markers: Protocols, applications,and overviews: (1997) 173-185, Cregan, et al., eds., Wiley-Liss N.Y.;all of which is herein incorporated by reference in its' entirely. Thehybridization of the probe to the target DNA molecule can be detected byany number of methods known to those skilled in the art, these caninclude, but are not limited to, fluorescent tags, radioactive tags,antibody based tags, and chemiluminescent tags.

Regarding the amplification of a target nucleic acid sequence (e.g., byPCR) using a particular amplification primer pair, “stringentconditions” are conditions that permit the primer pair to hybridize onlyto the target nucleic-acid sequence to which a primer having thecorresponding wild-type sequence (or its complement) would bind andpreferably to produce a unique amplification product, the amplicon, in aDNA thermal amplification reaction.

The term “specific for (a target sequence)” indicates that a probe orprimer hybridizes under stringent hybridization conditions only to thetarget sequence in a sample comprising the target sequence.

As used herein, “amplified DNA” or “amplicon” refers to the product ofnucleic-acid amplification of a target nucleic acid sequence that ispart of a nucleic acid template. For example, to determine whether thecorn plant resulting from a sexual cross contains transgenic eventgenomic DNA from the corn plant of the present invention, DNA extractedfrom a corn plant tissue sample may be subjected to nucleic acidamplification method using a DNA primer pair that includes a firstprimer derived from flanking sequence in the genome of the plantadjacent to the insertion site of inserted heterologous DNA, and asecond primer derived from the inserted heterologous DNA to produce anamplicon that is diagnostic for the presence of the event DNA. Theamplicon is of a length and has a sequence that is also diagnostic forthe event. The amplicon may range in length from the combined length ofthe primer pairs plus one nucleotide base pair, preferably plus aboutfifty nucleotide base pairs, more preferably plus about twohundred-fifty nucleotide base pairs, and even more preferably plus aboutfour hundred-fifty nucleotide base pairs. Alternatively, a primer paircan be derived from flanking sequence on both sides of the inserted DNAso as to produce an amplicon that includes the entire insert nucleotidesequence (e.g., the Mlu1 DNA fragment of the pMON25496 expressionconstruct, FIG. 1, approximately 6706 nucleotide base pairs). A memberof a primer pair derived from the plant genomic sequence may be locateda distance from the inserted DNA sequence, this distance can range fromone nucleotide base pair up to the limits of the amplification reaction,or about twenty thousand nucleotide base pairs. The use of the term“amplicon” specifically excludes primer dimers that may be formed in theDNA thermal amplification reaction.

Nucleic-acid amplification can be accomplished by any of the variousnucleic-acid amplification methods known in the art, including thepolymerase chain reaction (PCR). A variety of amplification methods areknown in the art and are described, inter alia, in U.S. Pat. Nos.4,683,195 and 4,683,202 and in PCR Protocols: A Guide to Methods andApplications, ed. Innis et al., Academic Press, San Diego, 1990. PCRamplification methods have been developed to amplify up to 22 kb ofgenomic DNA and up to 42 kb of bacteriophage DNA (Cheng et al., Proc.Natl. Acad. Sci. USA 91:5695-5699, 1994). These methods as well as othermethods known in the art of DNA amplification may be used in thepractice of the present invention. The sequence of the heterologous DNAinsert or flanking DNA sequence from corn event PV-ZMGT32(nk603) can beverified (and corrected if necessary) by amplifying such sequences fromDNA extracted from the ATCC deposit Accession No. PTA-2478 seed orplants using DNA primers derived from the sequences provided hereinfollowed by standard DNA sequencing of the PCR amplicon or of the clonedDNA.

The amplicon produced by these methods may be detected by a plurality oftechniques. One such method is Genetic Bit Analysis (Nikiforov, et al.Nucleic Acid Res. 22:41674175, 1994) where an DNA oligonucleotide isdesigned which overlaps both the adjacent flanking genomic DNA sequenceand the inserted DNA sequence. The oligonucleotide is immobilized inwells of a microwell plate. Following PCR of the region of interest(using one primer in the inserted sequence and one in the adjacentflanking genomic sequence), a single-stranded PCR product can behybridized to the immobilized oligonucleotide and serve as a templatefor a single base extension reaction using a DNA polymerase and labelledddNTPs specific for the expected next base. Readout may be fluorescentor ELISA-based. A signal indicates presence of the insert/flankingsequence due to successful amplification, hybridization, and single baseextension.

Another method is the Pyrosequencing technique as described by Winge(Innov. Pharma. Tech. 00:18-24, 2000). In this method an oligonucleotideis designed that overlaps the adjacent genomic DNA and insert DNAjunction. The oligonucleotide is hybridized to single-stranded PCRproduct from the region of interest (one primer in the inserted sequenceand one in the flanking genomic sequence) and incubated in the presenceof a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5′phosphosulfate and luciferin. DNTPs are added individually and theincorporation results in a light signal which is measured. A lightsignal indicates the presence of the transgene insert/flanking sequencedue to successful amplification, hybridization, and single or multi-baseextension.

Fluorescence Polarization as described by Chen, et al., (Genome Res.9:492-498, 1999) is a method that can be used to detect the amplicon ofthe present invention. Using this method an oligonucleotide is designedwhich overlaps the genomic flanking and inserted DNA junction. Theoligonucleotide is hybridized to single-stranded PCR product from theregion of interest (one primer in the inserted DNA and one in theflanking genomic DNA sequence and incubated in the presence of a DNApolymerase and a fluorescent-labeled ddNTP. Single base extensionresults in incorporation of the ddNTP. Incorporation can be measured asa change in polarization using a fluorometer. A change in polarizationindicates the presence of the transgene insert/flanking sequence due tosuccessful amplification, hybridization, and single base extension.

Taqman® (PE Applied Biosystems, Foster City, Calif.) is described as amethod of detecting and quantifying the presence of a DNA sequence andis fully understood in the instructions provided by the manufacturer.Briefly, a FRET oligonucleotide probe is designed which overlaps thegenomic flanking and insert DNA junction. The FRET probe and PCR primers(one primer in the insert DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermostable polymerase anddNTPs. Hybridization of the FRET probe results in cleavage and releaseof the fluorescent moiety away from the quenching moiety on the FRETprobe. A fluorescent signal indicates the presence of theflanking/transgene insert sequence due to successful amplification andhybridization.

Molecular Beacons have been described for use in sequence detection asdescribed in Tyangi, et al. (Nature Biotech. 14:303-308, 1996) Briefly,a FRET oligonucleotide probe is designed that overlaps the flankinggenomic and insert DNA junction. The unique structure of the FRET proberesults in it containing secondary structure that keeps the fluorescentand quenching moieties in close proximity. The FRET probe and PCRprimers (one primer in the insert DNA sequence and one in the flankinggenomic sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Following successful PCR amplification,hybridization of the FRET probe to the target sequence results in theremoval of the probe secondary structure and spatial separation of thefluorescent and quenching moieties. A fluorescent signal results. Afluorescent signal indicates the presence of the flanking/transgeneinsert sequence due to successful amplification and hybridization.

The following examples are included to demonstrate examples of certainpreferred embodiments of the invention. It should be appreciated bythose of skill in the art that the techniques disclosed in the examplesthat follow represent approaches the inventors have found function wellin the practice of the invention and thus can be considered toconstitute examples of preferred modes for its practice. However, thoseof skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentsthat are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

EXAMPLES Example 1

The PV-ZMGT32(nk603) (hence forth referred to as nk603) transgenic cornevent was generated by microprojectile bombardment of maize embryos(Songstad et al., In Vitro Cell Plant 32:179-183, 1996) using a linearMlu1 DNA fragment derived from pMON25496 (FIG. 1). This DNA fragmentcontains two transgene expression cassettes that collectively confercorn plant tolerance to glyphosate. The first cassette is composed ofthe rice actin 1 promoter and intron (P-Os.Act1 and I-Os.Act1, U.S. Pat.No. 5,641,876), operably connected to an Arabidopsis EPSPS chloroplasttransit peptide (TS-At.EPSPS:CTP2, Klee et al., Mol. Gen. Genet.210:47-442, 1987), operably connected to a glyphosate tolerant5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacteriumsp. strain CP4 (AGRTU.aroA:CP4, U.S. Pat. No. 5,633,435) and operablyconnected to a nopaline synthase transcriptional terminator(T-AGRTU.nos, Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803-4807,1983). The second transgene expression cassette consists of thecauliflower mosaic virus 35S promoter containing a tandem duplication ofthe enhancer region (P-CaMV.35S, Kay et al. Science 236:1299-1302, 1987;U.S. Pat. No. 5,164,316), operably connected to a Zea mays Hsp70 intron(I-Zm.Hsp70, U.S. Pat. No. 5,362,865), operably connected to anArabidopsis EPSPS chloroplast transit peptide (TS-At.EPSPS:CTP2, Klee etal., Mol. Gen. Genet. 210:47442, 1987), operably connected to aglyphosate tolerant 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS)from Agrobacterium sp. strain CP4 (AGRTU.aroA:CP4, U.S. Pat. No.5,633,435) and operably connected to a nopaline synthase transcriptionalterminator (T-AGRTU.nos, Fraley et al. Proc. Natl. Acad. Sci. USA80:4803-4807, 1983). Post-bombardment, glyphosate-tolerant transgeniccalli were selected on media containing 3 mM glyphosate and plants weresubsequently regenerated. Three hundred-four plants from 91 independenttransgenic events were produced, nk603 was selected from this populationbased on a superior combination of characteristics including glyphosatetolerance, agronomic performance, and single transgenic insertion.Greenhouse and field evaluations of the nk603 event and its derivedprogeny indicated this transgenic insertion confers tolerance thatexceeds commercial specifications for full vegetative and reproductivetolerance to 340 g glyphosate/acre (840 g glyphosate/hectare; 32 ozRoundup Ultra/acre) when applied at the V4 and V8 leaf stages.

Example 2

The glyphosate tolerant nk603 corn event was compared to the currentcommercial standard, GA21 (U.S. Pat. No. 6,040,497), for tolerance toglyphosate vegetative injury and effect on yield. GA21 contains at least3 transgene expression cassettes arranged in tandem in the genome ofGA21 event (SCP/GMO/232-Final, European Commission Health & ConsumerProtection Directorate-General). The transgene cassette of GA21 consistsof a rice actin 1 promoter and intron linked to a ribulose1,5-bisphosphate carboxylase chloroplast transit peptide linked to amodified maize glyphosate resistant EPSPS and a 3′ nopaline synthasetranscription termination region. Plants of nk603 and GA21 were plantedin rows in replicated field plots. The treatments were 1) unsprayed, 2)sprayed at a 64 ounces/acre rate of Roundup Ultra®(at V4 leaf stage andagain with 64 ounces/acre Roundup Ultra®(at V8 leaf stage, 3) sprayed ata 96 ounces/acre rate of Roundup Ultra® at V4 leaf stage and again with96 ounces/acre Roundup Ultra® at V8 leaf stage. Vegetative tolerance wasmeasured as percent vegetative injury determined by the amount of leafmalformation observed 10 days after the herbicide treatment at the V8leaf stage. The yield of each plot was measured in bushels/acre and thepercent reduction in yield determined for each herbicide treatmentrelative to the unsprayed treatment. The results shown in Table 1illustrates that nk603 shows a lower percent vegetative injury levelthan the GA21 plants and the observed percent yield reduction is alsoless for the nk603 event. A low incidence of vegetative injury wasobserved in the unsprayed plots, this observation is attributable tovarious environmental factors other than glyphosate herbicide exposure.The double expression cassette of pMON25496 in nk603 was compared tovegetative injury and fertility rating of 3 independent corn eventsobtained from only the CaMV.35S promoter driving expression of theglyphosate tolerance gene (AGRTU.aroA:CP4). It was observed that thedouble expression cassette conferred a higher level of vegetativetolerance and reproductive tolerance than three independent corn events(ev 1, ev 2, and ev 3) containing only the expression cassette where theglyphosate tolerance gene expression was driven by the CaMV.35Spromoter. A higher level of vegetative tolerance to glyphosate herbicideinjury was observed for nk603 plus 3 additional corn events derived frompMON25496 when compared to the average injury of 6 corn events derivedfrom a construct where the glyphosate tolerance gene expression wasdriven only by the rice actin promoter and intron (P-Os.Act1/I-Os.Act1).The plants transformed with the double expression cassette possessed ahigher level of glyphosate tolerance to vegetative and fertility injurythan plants derived from transformation with the single expressioncassettes this resulted in improved resistance to yield loss due toglyphosate herbicide application. The pMON25496 construct provides twoplant expression cassettes at a single location in nk603 that confers ahigher lever of glyphosate tolerance than the triple tandem insertionoccurring in the commercial standard, GA21. TABLE 1 Glyphosate Toleranceof nk603-Vegetative injury, Yield, and Fertility Rating % Veg Yield %Yield Event Treatment injury* (bushels/acre) red. GA21 Unsprayed 0.3142.2 64 oz Roundup Ultra ® at 5.3 134.1 5.7 V4 followed by 64 oz at V896 oz Roundup Ultra ® at 8.3 129.1 9.2 V4 followed by 96 oz at V8 nk603Unsprayed 0.9 145.6 64 oz Roundup Ultra ® at 2.9 138.5 4.9 V4 followedby 64 oz at V8 96 oz Roundup Ultra ® at 4.7 140.1 3.8 V4 followed by 96oz at V8 Fertility Treatment Rating** nk603 64 oz Roundup ® Ultra at V84.5 CaMV.35S ev 1 64 oz Roundup ® Ultra at V8 2.0 CaMV.35S ev 2 64 ozRoundup ® Ultra at V8 2.2 CaMV.35S ev 3 64 oz Roundup ® Ultra at V8 2.4ave. % Veg Treatment injury* nk603 plus 3 additional 128 oz Roundup ®Ultra at V4 22.9 pMON25496 events followed by 128 oz at V8 Six P—Os.Act1128 oz Roundup ® Ultra at V4 28.9 single cassette events followed by 128oz at V8*Veg injury is observed 10 days after V8 treatment is one measure takento assess vegetative injury in response to glyphosate treatment.**Male fertility rating: 4-5 = fully fertile; 3 = significantly reducedpollen shed; 0-2 = completely sterile-highly sterile, not suitable forcommercial use.

Example 3

The corresponding flanking DNA molecule from nk603 was cloned by usingligated adapters and nested PCR as described in the Genome Walker™ kit(catalog #K1807-1, CloneTech Laboratories, Inc, Palo Alto, Calif.).First, genomic DNA from the nk603 event was purified by the CTABpurification method (Rogers et al., Plant Mol. Biol. 5:69-76, 1985). Thegenomic DNA libraries for amplification were prepared according tomanufacturer's instructions (Genome Walker™, CloneTech Laboratories,Inc, Palo Alto, Calif.). In separate reactions, genomic DNA was digestedovernight at 37° C. with the following blunt-end restrictionendonucleases: EcoRV, ScaI, DraI, PvuII, and StuI (CloneTechLaboratories, Inc, Palo Alto, Calif.). The reaction mixtures wereextracted with phenol:chloroform, the DNA was precipitated by theaddition of ethanol to the aqueous phase, pelleted by centrifugation,then resuspended in Tris-EDTA buffer (10 mM Tris-.HCl, pH 8.0, 1 mMEDTA). The purified blunt-ended genomic DNA fragments were ligated tothe Genome Walker™ adapters according to the manufacturer's protocol.After ligation, each reaction was heat treated (70° C. for 5 min) toterminate the reaction and then diluted 10-fold in Tris-EDTA buffer. Oneμl of each respective ligation was then amplified in a 50 μM reactionthat included 1 μl of respective adapter-ligated library, 1 μl of 10 μMGenome Walker™ adapter primer AP1 (5′GTATATCGACTCACTATAGGGC 3′, SEQ IDNO: 1), 1 μl of 10 μM nk603 transgene-specific oligonucleotide (5′TGACGTATCAAAGTACCGACAAAAACATCC 3′ SEQ ID NO:2), 1 μl 10 mMdeoxyribonucleotides, 2.5 μl dimethyl sulfoxide, 5 μl of 10×PCR buffercontaining MgCl₂, 0.5 μl (2.5 units) of Amplitaq thermostable DNApolymerase (PE Applied Biosystems, Foster City, Calif.), and H₂O to 50μl. The reactions were performed in a thermocycler using calculatedtemperature control and the following cycling conditions: 1 cycle of 95°C. for 9 min; 7 cycles of 94° C. for 2 s, 70° C. for 3 min; 36 cycles of94° C. for 2 s, 65° C. for 3 min; 1 cycle of 65° C. for 4 min. One μl ofeach primary reaction was diluted 50-fold with water and amplified in asecondary reaction (1 μl of respective diluted primary reaction, 1 μl of10 μM Genome Walker™ nested adapter primer AP2 (5′ACTATAGGGCACGCGTGGT3′, SEQ ID NO:3, supplied by manufacturer), 1 μl of 10 μM nk603transgene-specific nested oligonucleotide(5′CTTTGTTTATTTTGGACTATCCCGACTC 3′, SEQ ID NO:4), 1 μl 10 mMdeoxyribonucleotides, 2.5 RI dimethyl sulfoxide, 5 μl of 10×PCR buffercontaining MgCl₂, 0.5 μl (2.5 units) of Amplitaq thermostable DNApolymerase (PE Applied Biosystems, Foster City, Calif.), and H₂O to 50μl] using the following cycling conditions: 1 cycle of 95° C. for 9 min;5 cycles of 94° C. for 2 s, 70° C. for 3 min; 24 cycles of 94° C. for 2s, 65° C. for 3 min; 1 cycle of 65° C. for 4 min.

PCR products, representing 5′ regions that span the junction between thenk603 transgenic insertion and the neighboring flanking corn DNA werepurified by agarose gel electrophoresis followed by purification fromthe agarose matrix using the QIAquick Gel Extraction Kit (catalog#28704, Qiagen Inc., Valencia, Calif.) and direct cloning into thepGEM-T Easy vector (catalog. # A1360, Promega, Madison, Wis.). Theidentity of the cloned PCR products and relationship to the Mlu Ifragment of pMON25496 was confirmed by DNA sequence analysis (ABI Prism377, PE Biosystems, Foster City, Calif. and DNASTAR sequence analysissoftware, DNASTAR Inc., Madison, Wis.).

Similarly, the nk603 3′ flanking corn DNA sequence was amplified andcloned using nested gene specific primers, such as, SEQ ID NO:5 (5′AGATTGAATCCTGTTGCCGGTCTTGC 3′) and SEQ ID NO:6 (5′GCGGTGTCATCTATGTTACTAGATCGGG 3′) that anneal to the T-AGRTU.nostranscriptional terminator. Two T-AGRTU.nos transcriptional terminatorsare present in the nk603 transgenic/genomic insertion, one internal inthe construct and one at the 3′ end of the construct adjacent to corngenomic DNA. The PCR products produced in this reaction were sequencedand the DNA sequence that spans the junction between transgene andflanking sequence was distinguished from products of the internalT-AGRTU.nos by comparison to the known genetic element sequences of thepMON25496 construct as previously described.

Corn DNA sequence flanking both sides of the transgenic insertion wasdetermined for nk603 by sequencing the Genome Walker™-derivedamplification products and alignment to known transgene sequence. At the5′ end of the transgenic insertion, the sequence of a 498 bp segmentaround the insertion junction was determined (SEQ ID NO:7). Thisconsisted of 304 base pairs (bp) of the flanking maize genomic DNAsequence (nucleotides 1-304 of SEQ ID NO:7), 45 bp of pMON25496construct DNA sequence (nucleotides 305-349 of SEQ ID NO:7) and 149 bpof DNA sequence from the 5′ end of P-Os.Act1 (nucleotides 350-498 of SEQID NO:7).

The DNA sequence was determined for a 1183 bp segment around the 3′insertion junction (SEQ ID NO:8), that begins with 164 bp of theT-AGRTU.nos transcriptional terminator (nucleotides 1-164 of SEQ IDNO:8), 217 bp of pMON25496 construct DNA sequence (nucleotides 165-381of SEQ ID NO:8), 305 bp of the maize plastid genes, rpsll and rpoA(partial segments of each gene corresponding to bases 63-363 of Genbankaccession X07810, corresponding to bases 382-686 of SEQ ID NO:8), andthe remaining DNA sequence consisting of maize genomic DNA sequenceflanking the integration site (corresponding to bases 687-1183 of SEQ IDNO:8).

The junction DNA molecules, SEQ ID NO:9, 10, 11, and 12 are novel DNAmolecules in nk603 and are diagnostic for corn plant nk603 and itsprogeny. The junction molecules in SEQ ID NO:9, 10, and 11 representabout 9 nucleotides on each side of an insertion site of a transgene DNAfragment and corn genomic DNA. SEQ ID NO:9 is found at nucleotidepositions 295-314 of SEQ ID NO:7. The junction sequences SEQ ID NO: 10and 11 are located at nucleotide positions 373-390 and 678-695,respectively, of SEQ ID NO:8, representing a junction DNA molecule ofconstruct DNA sequence with corn plastid DNA sequence (SEQ ID NO:10) andconstruct sequence with corn genomic DNA sequence (SEQ ID NO: 11). SEQID NO: 12 is located at nucleotide position 156-173 of SEQ ID NO:8 andrepresents a novel DNA molecule in nk603 due to it being a fusion of theT-AGRTU.nos terminator sequence with an inverted fragment of the riceactin promoter DNA sequence.

Example 4

DNA event primer pairs are used to produce an amplicon diagnostic fornk603. These event primer pairs include but are not limited to SEQ IDNO: 13 and SEQ ID NO: 14 for the 5′ amplicon DNA molecule and SEQ ID NO:15 and SEQ ID NO: 16 for the 3′ amplicon DNA molecule. In addition tothese primer pairs, any primer pair derived from SEQ ID NO:7 and SEQ IDNO:8 that when used in a DNA amplification reaction produces a DNAamplicon diagnostic for nk603 is an aspect of the present invention. Theamplification conditions for this analysis is illustrated in Table 2 andTable 3 for the 5′ transgene insert/genomic junction region. The samemethod is applied for amplification of the 3′ amplicon DNA moleculeusing primer DNA molecules SEQ ID NO: 15 and SEQ ID NO: 16, however, anymodification of these methods that use DNA molecules or complementsthereof to produce an amplicon DNA molecule diagnostic for nk603 iswithin the ordinary skill of the art. In addition, a control primer pair(SEQ ID NO:17 and 18) for amplification of an endogenous corn gene isincluded as an internal standard for the reaction conditions. Theanalysis of nk603 plant tissue DNA extract sample should include apositive tissue DNA extract control from nk603, a negative DNA extractcontrol from a corn plant that is not nk603, and a negative control thatcontains no template corn DNA extract. Additional DNA primer moleculesof sufficient length can be selected from SEQ ID NO:7 and SEQ ID NO:8 bythose skilled in the art of DNA amplification methods, and conditionsoptimized for the production of an amplicon that may differ from themethods shown in Table 2 and Table 3 but result in an amplicondiagnostic for nk603. The use of these DNA primer sequences withmodifications to the methods of Table 2 and 3 are within the scope ofthe invention. The amplicon wherein at least one DNA primer molecule ofsufficient length derived from SEQ ID NO:7 and SEQ ID NO:8 that isdiagnostic for nk603 is an aspect of the invention. The amplicon whereinat least one DNA primer of sufficient length derived from any of thegenetic elements of pMON25496 that is diagnostic for nk603 is an aspectof the invention. The assay for the nk603 amplicon can be performed byusing a Stratagene Robocycler, MJ Engine, Perkin-Elmer 9700, orEppendorf Mastercycler Gradient thermocycler as shown in Table 3, or bymethods and apparatus known to those skilled in the art. TABLE 2 PCRprocedure and reaction mixture for the confirmation of nk603 5′transgene insert/genomic junction region. Step Reagent Amount Comments 1Nuclease-free water add to final — volume of 20 μl 2 10× reaction 2.0 μl1× final buffer (with MgCl₂) concentration of buffer, 1.5 mM finalconcentration of MgCl₂ 3 10 mM solution 0.4 μl 200 μM final of dATP,dCTP, concentration of dGTP, and dTTP each dNTP 4 event primer 0.4 μl0.2 μM final (SEQ ID NO: 13) concentration (resuspended in 1× TE bufferor nuclease-free water to a concentration of 10 μM) 5 event primer 0.4μl 0.2 μM final (SEQ ID NO: 14) concentration (resuspended in 1× TEbuffer or nuclease-free water to a concentration of 10 μM) 6 controlprimer 0.2 μl 0.1 μM final (SEQ ID NO: 17) concentration (resuspended in1× TE buffer or nuclease-free water to a concentration of 10 μM) 7control primer 0.2 μl 0.1 μM final (SEQ ID NO: 18) concentration(resuspended in 1× TE buffer or nuclease-free water to a concentrationof 10 μM) 8 RNase, DNase 0.1 μl 50 ng/reaction free (500 ng/μl) 9 REDTaqDNA 1.0 μl 1 unit/reaction polymerase (recommended (1 unit/μl) to switchpipets prior to next step) 10 Extracted DNA — (template): Samples to beanalyzed individual 10-200 ng of leaves genomic DNA pooled leaves 200 ngof (maximum of genomic DNA 50 leaves/pool) Negative 50 ng of corncontrol genomic DNA (notnk603) Negative no template DNA control Positive50 ng of nk603 control genomic DNA

TABLE 3 Suggested PCR parameters for different thermocyclers Gently mixand, if needed (no hot top on thermocycler), add 1-2 drops of mineraloil on top of each reaction. Proceed with the PCR in a StratageneRobocycler, MJ Engine, Perkin-Elmer 9700, or Eppendorf MastercyclerGradient thermocycler using the following cycling parameters. Cycle No.Settings: Stratagene Robocycler 1 94° C. 3 minutes 38  94° C. 1 minute60° C. 1 minute 72° C. 1 minute and 30 seconds 1 72° C. 10 minutes CycleNo. Settings: MJ Engine or Perkin-Elmer 9700 1 94° C. 3 minutes 38  94°C. 10 seconds 60° C. 30 seconds 72° C. 1 minute 1 72° C. 10 minutesCycle No. Settings: Eppendorf Mastercycler Gradient 1 94° C. 3 minutes38  94° C. 15 seconds 60° C. 15 seconds 72° C. 1 minute 1 72° C. 10minutesNote:The MJ Engine or Eppendorf Mastercycler Gradient thermocycler should berun in the calculated mode. Run the Perkin-Elmer 9700 thermocycler withthe ramp speed set at maximum.

A deposit of the Monsanto Company, corn seed of event PV-ZMGT32(nk603)disclosed above has been made under the Budapest Treaty with theAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110. The ATCC accession number is PTA-2478. The depositwill be maintained in the depository for a period of 30 years, or 5years after the last request, or for the effective life of the patent,whichever is longer, and will be replaced as necessary during thatperiod.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

All publications and published patent documents cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

1. A DNA construct comprising: a first and a second expression cassette,wherein said first expression cassette in operable linkage comprises (i)a rice actin 1 promoter; (ii) a rice actin 1 intron; (iii) a chloroplasttransit peptide encoding DNA molecule; (iv) a glyphosate tolerant EPSPSencoding DNA molecule; and (v) a transcriptional terminator DNAmolecule; and said second expression cassette comprising in operablelinkage (a) a CaMV 35S promoter; (b) a Hsp70 intron; (c) a chloroplasttransit peptide encoding DNA molecule; (d) a glyphosate tolerant EPSPSencoding DNA molecule; and (e) a transcriptional terminator DNAmolecule.
 2. A DNA construct of claim 1, wherein the glyphosate tolerantEPSPS encoding DNA molecule consists of an AGRTU.aroA:CP4 DNA molecule.3. A plant comprising the DNA construct of claim
 2. 4. A plant of claim3, wherein said plant is a corn plant.
 5. A corn plant provided by ATCCseed deposit PTA-2478, progeny seeds and plants or parts thereof.
 6. ADNA molecule comprising a nucleotide sequence identified as SEQ ID NO:7or SEQ ID NO:8.
 7. A pair of DNA molecules comprising: a first DNAmolecule and a second DNA molecule, wherein the DNA molecules are ofsufficient length of contiguous nucleotides of SEQ ID NO:7 or itscomplement to function as DNA primers or probes diagnostic for DNAextracted from corn plant PV-ZMGT32(nk603) or progeny thereof.
 8. A pairof DNA molecules comprising: a first DNA molecule and a second DNAmolecule, wherein the DNA molecules are of sufficient length ofcontiguous nucleotides of SEQ ID NO:8 or its complement to function asDNA primers or probes diagnostic for DNA extracted from corn plantPV-ZMGT32(nk603) or progeny thereof.
 9. A method of detecting thepresence of a DNA molecule in a corn plant provided by ATCC seed depositPTA-2478 or progeny thereof, the method comprising: (a) extracting a DNAsample from said corn plant provided by ATCC seed deposit PTA-2478 orprogeny seeds and plants or parts thereof; (b) contacting the DNA samplewith a DNA primer pair comprising DNA primer molecules of sufficientlength of contiguous nucleotides of SEQ ID NO:7 or its complement, orSEQ ID NO:8 or its complement; (c) providing a nucleic acidamplification reaction condition; (d) performing said nucleic acidamplification reaction, thereby producing a DNA amplicon moleculecomprising the DNA molecules selected from the group consisting of SEQID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12; and (e) detectingthe DNA amplicon molecule.
 10. A corn plant when analyzed by a method ofclaim 9 produced an amplicon comprising the DNA molecules selected fromthe group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQID NO:12.
 11. In the method of claim 9, the DNA amplicon moleculecomprising the DNA molecules selected from the group consisting of SEQID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.
 12. A method ofdetecting the presence of a DNA molecule selected from the groupconsisting of SEQ ID NO:7 and SEQ ID NO:8 in a DNA sample, the methodcomprising: (a) extracting a DNA sample from a corn plant; (b)contacting the DNA sample with a DNA molecule identified as SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, wherein said DNA moleculeis a DNA probe that hybridizes under stringent hybridization conditionswith the DNA molecule selected from the group consisting of SEQ ID NO:7or SEQ ID NO:8, and does not hybridize under the stringent hybridizationconditions with a DNA sample not containing the DNA molecule identifiedas SEQ ID NO:7 or SEQ ID NO:8; (c) subjecting the sample and probe tostringent hybridization conditions; and detecting hybridization of theprobe to the DNA.
 13. A DNA molecule selected from the group consistingof SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, andcomplements thereof.
 14. A method of breeding a glyphosate toleranttrait into corn plants comprising a) extracting a DNA sample fromprogeny corn plants; b) contacting the DNA sample with a marker nucleicacid molecule selected from the group consisting of SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, and complements thereof. c)performing a marker assisted breeding method for the glyphosate toleranttrait, wherein the glyphosate tolerant trait is genetically linked to acomplement of the marker nucleic acid molecule;
 15. A method ofproducing a corn plant that tolerates application of glyphosateherbicide comprising: (a) transforming a corn cell with the DNAconstruct of claim 1; (b) selecting said corn cell for tolerance toapplication of glyphosate; (c) growing said corn cell into a fertilecorn plant;
 16. A DNA detection kit comprising: at least one DNAmolecule of sufficient length of contiguous nucleotides homologous orcomplementary to SEQ ID NO:7 or SEQ ID NO:8 that functions as a DNAprimer or probe specific for corn event PV-ZMGT32(nk603) and itsprogeny.